Protein RPE65 plays a role in recycling of 11-cis retinal and thus, is essential for regeneration of visual pigments in photoreceptors. Defects in RPE65 genes are associated with several forms of retinal dystrophies. However, the function of RPE65 is presently unknown. As a result, the pathogenesis of the retinal degeneration resulting from RPE65 defects has not been elucidated. In previous study, we have shown that RPE65 is essential for isomerization of all-trans retinoids to generate 11-cis isomers. Several lines of evidence suggest that RPE65 associates with other retinoid-processing proteins to form a multi-enzyme complex. This project proposes to identify the components of this complex, i.e., partners of RPE65, by two strategies: 1) cross-linking and co-purification, and 2) yeast two-hybrid system. This study will reveal the structure of this important complex and provide new insights into RPE65 function and its interactions with other retinoid-processing enzymes. Multiple point mutations have been identified in the RPE65 gene from patients with retinal dystrophies. We hypothesize that these mutations impair the isomerase activity. Our recent results showed that recombinant RPE65 restored the isomerase activity in the RPE from RPE65-/- mice. Using this assay system, we will evaluate the impacts of the RPE65 mutations on the isomerase activity. This approach will contribute to the identification of key residues for RPE65 function and to the elucidation of the structure of this important protein. Defects in RPE65 cause interruption of the visual cycle of retinoids and may alter the expression of multiple genes, which contribute to retinal degenerations. Using the microarray technique, we propose to identify genes with changed expression in RPE65-/- mice. This approach may identify some retinoid-processing proteins and thus, contribute to the delineation of retinoid metabolic pathways. It has potential to identify some apoptotic genes that may be responsible for the retinal degeneration in the knock-out mouse. Toward the understanding of pathogenesis of retinal degeneration caused by RPE65 defects, we propose to examine the effect of the interrupted visual cycle, especially the accumulation of retinyl ester, on functions of the RPE and the impacts on photoreceptor integrity. Ester accumulation will be prevented by vitamin A deprivation in the knock-out mice, and consequent time courses of photoreceptor degeneration and ester accumulation will be compared with their littermates on a regular diet. The results will determine whether the lack of 11-cis retinal or accumulation of ester is responsible for the retinal degeneration. These studies may generate useful information for understanding of inherited retinal degenerations and have impacts on the treatment of these blinding diseases.